Hematopoietic cells are believed to arise in the bone marrow from a totipotent stem cell. The stem cell is able to renew itself as well as to give rise to progenitor cells such as the erythroid progenitors and myeloid progenitors. The progenitor cells, in turn, give rise to differentiated cells which are morphologically recognizable as belonging to a certain lineage such as the erythroid, megakaryocytic, myeloid, and lymphoid lineages, and which have a limited or no capacity to proliferate. In humans, stem cells and progenitor cells express the CD34 antigen, while more differentiated hematopoietic cells do not.
Stem cells and progenitor cells do not execute their development programs autonomously. Activities produced in the marrow microenvironment signal the progenitor cells to divide and differentiate. Thus, defining the functional components of the bone marrow microenvironment is a prerequisite to understanding how the proliferation and differentiation of progenitor cells is coordinately regulated. The cellular complexity of the marrow microenvironment has been demonstrated both in situ and in vitro by a variety of histochemical techniques (Lichtman, Exp. Hematol. 9:391 (1981), and Allen et al., Exp. Hematol. 12:517 (1984)). The marrow microenvironment is comprised of both hematopoietic and stromal or mesenchymal derived cells. The stromal cells include endothelial cells that form the sinuses and adventitial reticular cells that have characteristics consistent with adipocytes, fibroblasts, and muscle cells (Charbord et al., Blood 66:1138 (1985), and Charbord et al., Exp. Hematol. 18:276 (1990)). Numerous advances in recent years have provided considerable information on the ontogeny and development of hematopoietic cells; however, ontogeny of the stromal components and their precise role in controlling hematopoiesis has proven elusive (Ogawa, Blood 81:2844 (1993); Muller-Sieburg et al., Critical Rev. Immunol. 13:115 (1993); and Dorshkind, Ann. Rev. Immunol. 8:111 (1990)).
Long term cultures of marrow cells are an in vitro approximation of the in vivo marrow microenvironment and have been informative with respect to the identification of growth factors, adhesion proteins and extracellular matrix proteins that mediate the interaction between the hematopoietic cells and the stromal elements (Muller-Sieburg et al., supra; Dorshkind, supra; Liesveld et al., Exp. Hematol. 9:391 (1981); Kittler et al., Blood 79:3168 (1992); Eaves et al., Blood 78:110 (1991); Clark et al., Bailliere's Clin. Haematol. 5:619 (1992); and Heinrich et al., Blood 82:771 (1993)). One improvement to this system was the use of stromal precursors, positive for the STRO-1 antigen, to initiate long term cultures (LTC); STRO-1 positive stromal precursors are devoid of myeloid components and less heterogeneous than primary cultures, but are still capable of supporting hematopoiesis (Simmons and Torok-Storb, Blood 78:55-62 (1991)). However, both the STRO-1 initiated cultures and the primary LTC are too complex to delineate contributions from individual cell types. Moreover, primary cultures can be highly variable and change with time, further complicating the identification of stromal cells that have a role in controlling hematopoiesis.
Immortalized stromal cell lines have been used to circumvent some of these problems. Numerous spontaneous murine cell lines have been established (Zipori et al., J. Cell Physiol. 118:143 (1984); Zipori et al., J. Cell Physiol. 122:81 (1985); and Song et al., Exp. Hematol. 12:523 (1984)), however, unlike mouse lines human cell lines undergo senescence unless first immortalized by transformation with a retrovirus (Lanotte et al., J. Cell Sci. 50:281 (1981)). The few human bone marrow stromal cell lines that are available were established using the SV40 virus large T antigen (Harigaya et al., Proc. Natl. Acad. Sci. USA 82:3477 (1985); Tsai et al., J. Cell Physiol. 127:137 (1986); Novotny et al., Exp. Hematol. 18:775 (1990); Slack et al., Blood 75:2319 (1990); Singer et al., Blood 70:464 (1987); Cicutinni et al., Blood 80:102 (1992); and Thalmeir et al., Blood 83:1799 (1994)). Some of these lines are promising with respect to the maintenance of hematopoietic cells; unfortunately, some also display transformed phenotypes which limits their usefulness for extrapolation to the normal marrow microenvironment (Novotony, supra).
The ability to culture hematopoietic cells and their precursors, derived from the bone marrow, peripheral blood, or umbilical cord blood of a patient or donor, offers the potential to overcome the disadvantages of immunosuppressive or immunodestructive therapies which are often used in the treatment of cancer and other life-threatening diseases. Cultured hematopoietic cells can be used as an important source of proliferating cells to reconstitute a patient's blood-clotting and infection-fighting functions subsequent to therapy. In addition, the ability to expand hematopoietic cells and their precursors in vitro may relieve dependence on bone marrow aspiration or multiple aphereses as the only means of obtaining sufficient cells for transplantation.
Early work in the field of hematopoietic stem cell culture centered around the culture of murine bone marrow aspirates in agar gel or liquid medium. Unfractionated bone marrow (including stem cells, progenitor cells, more differentiated hematopoietic cells, and stromal elements) was used to inoculate the cultures, but they were generally short-lived and resulted in little or no increase in cell number, particularly in the stem cell and progenitor compartments. The results were even less promising when human bone marrow was employed. The human cells generally adhered to the bottom and sides of the culture vessel and their removal was difficult.
Subsequent efforts focused on inoculating mouse bone marrow onto preestablished monolayers of bone marrow stromal cells (so-called Dexter cultures; Dexter, Acta Haematol, 62:299-305, 1979). While some success was obtained with Dexter cultures of mouse cells, the same approach was disappointing with human cells, in that a steady decline in the numbers of all cell types is observed in human Dexter cultures (Quesenberry, Curr. Topics Microbiol. Immunol. 177:151 (1992)).
A further disadvantage of Dexter cultures is that, to the extent that there is expansion of hematopoietic precursor cells, these cells adhere to the stromal layer and are extremely difficult to recover from the culture without employing conditions which damage the cells. The proliferating cells which are released into the culture medium (that is, the non-adherent cells) are generally more mature cells, which cannot restore sustained hematopoiesis in a transplanted individual.
There remains a need in the art for a method of culturing human hematopoietic cells, which method (a) results in expansion of the number of hematopoietic precursor cells; (b) enhances the yield and recovery of the precursor cells without compromising viability; and (c) can be independent of the presence of bone marrow stromal elements. Quite surprisingly, the present invention fulfills this and other related needs.